Replication stress pathway agent compositions and methods for treating cancer

ABSTRACT

Provided herein are methods of treating cancer in a subject, wherein the cancer is extrachromosomal DNA-positive (ecDNA-positive) or therapeutically resistant, the method comprising administering to the subject a therapeutically effective amount of a replication stress (RS) pathway agent alone or in combination with a targeted therapeutic.

CROSS REFERENCE

This patent application is a continuation of U.S. application Ser. No.17/568,434, filed Jan. 4, 2022, which is a continuation of internationalpatent application number PCT/US2021/045556, filed Aug. 11, 2021, whichclaims the benefit of U.S. Provisional Application No. 63/064,555, filedAug. 12, 2020, and U.S. Provisional Application No. 63/168,120, filedMar. 30, 2021, all of which are incorporated by reference herein intheir entirety.

BACKGROUND

Cancers often prove resistant to the therapeutics that are used to treatthem, frustrating efforts to extend progression free survival in cancerpatients. In some cases, treatment resistant cancers are observed to bepositive for extrachromosomal DNA (ecDNA), which sometimes containsamplified oncogenes, contributing to therapeutic resistance.

SUMMARY

In an aspect, there are provided methods for treating a tumor or tumorcells in a subject. In some embodiments, the method comprisesadministering a replication stress pathway agent (RSPA) in an amountsufficient to induce replication stress in the tumor or tumor cells; andadministering a cancer-targeted therapeutic agent, wherein the tumor ortumor cells have an ecDNA signature, and wherein growth or size of thetumor or growth or number of tumor cells is reduced. In someembodiments, the ecDNA signature is selected from the group consistingof a gene amplification; a p53 loss of function mutation; absence ofmicrosatellite instability (MSI-H); a low level of PD-L1 expression; alow level of tumor inflammation signature (TIS); a low level of tumormutational burden (TMB); an increased frequency of allele substitutions,insertions, or deletions (indels); and any combination thereof. In someembodiments, the gene amplification comprises an amplification of anoncogene, a drug-resistance gene, a therapeutic target gene, or acheckpoint inhibitor gene. In some embodiments, the cancer-targetedtherapeutic agent is directed to an activity of a protein product of atarget gene, and wherein the treatment with the cancer-targetedtherapeutic agent and the RSPA reduces amplification or expression ofthe target gene in the tumor or tumor cells. In some embodiments, thecancer-targeted therapeutic agent is administered prior to the RSPA. Insome embodiments, the tumor or tumor cells develop the ecDNA signatureafter administration of the cancer-targeted therapeutic agent. In someembodiments, the cancer-targeted therapeutic agent is administeredconcurrently with the RSPA. In some embodiments, the tumor or tumorcells develop the ecDNA signature prior to treatment. In someembodiments, the method prevents an increase of ecDNA in the tumor ortumor cells. In some embodiments, the cancer-targeted therapeutic agenttargets a protein product of an oncogene. In some embodiments, theoncogene comprises a point mutation, an insertion, a deletion, a fusion,or a combination thereof. In some embodiments, the cancer-targetedtherapeutic agent targets a gene selected from the group consisting ofABCB1, AKT, ALK, AR, BCL-2, BCR-ABL, BRAF, CDK4, CDK6, c-MET, EGFR. ER,ERBB3, ERRB2, AK, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GR, HRAS, IGF1R,KIT, KRAS, MCL-1, MDM2, MDM4, MTOR, MYC, MYCL, MYCN, NRAS, NRG1, NTRK1,NTRK2, NTRK3, PDGFR, PIK3Cδ, PIK3CA/B, RET, and ROS1. In someembodiments, the tumor or tumor cells comprise an amplification of afirst gene or portion thereof. In some embodiments, the first gene is anoncogene or a drug resistance gene. In some embodiments, theamplification is present on ecDNA. In some embodiments, the first geneis selected from the group consisting of ABCB1, AKT, ALK, AR, BCL-2,BCR-ABL, BRAF, CDK4, CDK6, c-MET, EGFR. ER, ERBB3, ERRB2, AK, FGFR1,FGFR2, FGFR3, FGFR4, FLT3, GR, HRAS, IGF1R, KIT, KRAS, MCL-1, MDM2,MDM4, MTOR, MYC, MYCL, MYCN, NRAS, NRG1, NTRK1, NTRK2, NTRK3, PDGFR,PIK3Cδ, PIK3CA/B, RET, and ROS1. In some embodiments, thecancer-targeted therapeutic agent is directed against the first gene. Insome embodiments, the subject has not been previously treated with thecancer-targeted therapeutic agent. In some embodiments, the tumor ortumor cells have not been previously treated with the cancer-targetedtherapeutic agent. In some embodiments, the method prevents an increaseof ecDNA in the tumor or tumor cells. In some embodiments, the tumor ortumor cells are resistant or non-responsive to a previous therapeuticagent prior to treatment with the cancer-targeted therapeutic agent andthe RSPA. In some embodiments, the tumor or tumor cells have beenpreviously treated with the previous therapeutic agent. In someembodiments, the subject has been previously treated with the previoustherapeutic agent. In some embodiments, the cancer-targeted therapeuticagent is directed to an activity of a protein product of a target gene,and wherein the treatment with the cancer-targeted therapeutic agent andthe RSPA reduces amplification or expression of the target gene in thetumor or tumor cells. In some embodiments, the target gene is anoncogene, a drug-resistance gene, a therapeutic target gene, or acheckpoint inhibitor gene. In some embodiments, the target gene isselected from the group consisting of ABCB1, AKT, ALK, AR, BCL-2,BCR-ABL, BRAF, CDK4, CDK6, c-MET, EGFR, ER, ERBB3, ERRB2, AK, FGFR1,FGFR2, FGFR3, FGFR4, FLT3, GR, HRAS, IGF1R, KIT, KRAS, MCL-1, MDM2,MDM4, MTOR, MYC, MYCL, MYCN, NRAS, NRG1, NTRK1, NTRK2, NTRK3, PDGFR,PIK3Cδ, PIK3CA/B, RET, and ROS1. In some embodiments, the ecDNAsignature is known prior to beginning treatment of the tumor or tumorcells. In some embodiments, the ecDNA signature is known after beginningtreatment of the tumor or tumor cells. In some embodiments, the methodimproves an objective response rate and/or extends a duration oftreatment response as compared to treatment with the cancer-targetedtherapeutic agent in the absence of the RSPA. In some embodiments, themethod increases a period of progression free survival as compared totreatment with the cancer-targeted therapeutic agent in the absence ofthe RSPA.

In another aspect, there are provided methods of treating anecDNA-associated tumor or tumor cells comprising administering a RSPAand a cancer-targeted therapeutic agent to a subject identified ashaving a tumor or tumor cells having ecDNA, wherein growth or size ofthe tumor or growth or number of the tumor cells is decreased as aresult of treatment. In some embodiments, the tumor or tumor cells ofthe subject are identified as having an ecDNA signature. In someembodiments, the ecDNA signature is selected from the group consistingof a gene amplification; a p53 loss of function mutation; absence ofmicrosatellite instability (MSI-H); a low level of PD-L1 expression; alow level of tumor inflammation signature (TIS); a low level of tumormutational burden (TMB); an increased frequency of allele substitutions,insertions, or deletions (indels); and any combination thereof. In someembodiments, the gene amplification comprises amplification of anoncogene, a drug-resistance gene, a therapeutic target gene, or acheckpoint inhibitor gene. In some embodiments, the tumor or tumor cellsare identified as having ecDNA by imaging ecDNA in cells, detectingecDNA using an oncogene binding agent, or by DNA sequencing. In someembodiments, ecDNA is identified in circulating tumor DNA.

In various aspects of methods herein, in some embodiments, the tumor ortumor cells are comprised by a solid tumor. In some embodiments,presence of ecDNA in the solid tumor is reduced or abolished as a resultof treatment. In some embodiments, a level of ecDNA is reduced in thesolid tumor after treatment as compared to the level of ecDNA prior totreatment. In some embodiments, a level of oncogene amplification and/ora level of copy number variation (CNV) in the solid tumor is reducedafter treatment as compared to the level of oncogene amplificationand/or CNV in the solid tumor prior to treatment. In some embodiments,the tumor or tumor cells include circulating tumor cells. In someembodiments, presence of ecDNA in the circulating tumor cells is reducedor abolished as a result of treatment. In some embodiments, a level ofecDNA is reduced in the circulating tumor cells after treatment ascompared to the level of ecDNA prior to treatment. In some embodiments,a level of oncogene amplification and/or a level of copy numbervariation (CNV) in the circulating tumor cells is reduced aftertreatment as compared to the level of oncogene amplification and/or CNVin the circulating tumor cells prior to treatment. In some embodiments,the presence or level of ecDNA is identified in circulating tumor DNA.In some embodiments, the RSPA is selected from the group consisting of aRNR inhibitor, an ATR inhibitor, a CHK1 inhibitor, a WEE1 inhibitor, anda PARG inhibitor. In some embodiments, the RNR inhibitor is selectedfrom the group consisting of gemcitabine, hydroxyurea, triapine,5-chloro-2-(n-((1S,2R)-2-(6-fluoro-2,3-dimethylphenyl)-1-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)propyl)sulfamoyl)benzamide,clofarabine, fluarabine, motexafin gadolinium, cladribine, tezacitabine,and COH29(N-[4-(3,4-dihydroxyphenyl)-5-phenyl-1,3-thiazol-2-yl]-3,4-dihydroxybenzamide).In some embodiments, the CHK1 inhibitor is selected from the groupconsisting of GDC-0575, prexasertib, LY-2880070, SRA737, XCCS-605B,rabusertib (LY-2603618), SCH-900776, RG-7602, AZD-7762, PF-477736, andBEBT-260. In some embodiments, the WEE1 inhibitor is selected from thegroup consisting of AZD1775 (MK1775), ZN-c3, Debio 0123, IMP7068,SDR-7995, SDR-7778. NUV-569, PD0166285, PD0407824, SC-0191, DC-859/A,bosutinib, and Bos-I. In some embodiments, the ATR inhibitor is selectedfrom the group consisting of RP-3500, M-6620, berzosertib (M-6620,VX-970; VE-822), AZZ-6738, AZ-20, M-4344 (VX-803), BAY-1895344, M-1774,IMP-9064, nLs-BG-129, SC-0245, BKT-300, ART-0380, ATRN-119, ATRN-212,NU-6027. In some embodiments, the cancer targeted therapeutic agent isselected from the group consisting of abemaciclib, ado-trastuzumabemtansine, afatinib, alectinib, ALRN-6924, AMG232, AMG-510, apatinib,ARS-3248, AXL1717, bevacizumab, bortezomib, brigatinib, cabozantinib,capmatinib, ceritinib, cetuximab, CGM097, crizotinib, crizotinib,dabrafenib, dacomitinib, dasatinib, doxorubicin, DS-3032b, encorafenib,entrectinib, erdafitinib, erlotinib, everolimus, fam-trastuzumabderuxtecan, figitumumab, gefitinib, gossypol, HDM201, idasanutlin,imatinib, infigratinib, iniparib, lapatinib, larotrectinib, LEE011,lenvatinib, LGX818, lorlatinib, MEK162, MK-8242 (SCH-900242), MRTX849,navitoclax, necitumumab, nilotinib, obatoclax, olaparib, OSI-906,osimertinib, palbociclib, panitumumab, PD-0332991, perisofine,pertuzumab, PL225B, repotrectinib, ribociclib, RO5045337, salinomycin,salirasib, SAR405838 (MI-77301), sorafenib, sotorasib, sunitinib,tamoxifen, temsirolimus, tipifanib, tivanitab, tofacitinib, trametinib,trastuzumab, tucatinib, UPR1376, VAL-083, vemurafenib, vemurafenib,vintafolide, and zoptarelin. In some embodiments, the RSPA is an RNRinhibitor and the RSPA is administered at a sub-therapeutic doserelative to its recommended use as a single agent. In some embodiments,the RNR inhibitor is gemcitabine. In some embodiments, the RNR inhibitoris not gemcitabine or hydroxyurea. In some embodiments, the RSPA is notgemcitabine. In some embodiments, the RSPA is not gemcitabine when thecancer-targeted therapeutic agent is an EGFR inhibitor.

In an aspect, there are provided methods for treating cancer in asubject in need thereof. In some cases, the method comprises:administering to the subject a therapeutically effective amount of areplication stress (RS) pathway inhibitor, (also referred to herein as areplication stress pathway agent or RSPA), wherein the cancer has beendetermined to be extrachromosomal DNA-positive (ecDNA-positive). In somecases, the RS pathway inhibitor comprises a RNR inhibitor, an ATRinhibitor, a CHK1 inhibitor, an E2F inhibitor, an WEE1 inhibitor, a PARGinhibitor, or a RRM2 inhibitor. In some cases, the RNR inhibitorcomprises Gemcitabine, hydroxyurea, triapine, or5-chloro-2-(n-((1S,2R)-2-(6-fluoro-2,3-dimethylphenyl)-1-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)propyl)sulfamoyl)benzamide.In some cases, the CHK1 inhibitor comprises GDC-0575, prexasertib, orSRA737. In some cases, the ecDNA-positive cancer comprises an amplifiedoncogene on the ecDNA. In some cases, the oncogene comprises one or moreof BRAF, CCND1, CDK4, CDK6, c-Myc, EGFR, ERB2, FGFR, HRAS, IGF1R, KRAS,MDM2, MDM4, MET, MYCL, MYCN, and NRAS. In some cases, the method furthercomprises administering to the subject a therapeutically effectiveamount of a targeted therapeutic that inhibits the protein product ofthe amplified oncogene. In some cases, the targeted therapeuticcomprises abemaciclib, ado-trastuzumab emtansine, afatinib, alectinib,ALRN-6924, AMG232, AMG-510, apatinib, ARS-3248, AXL1717, AZD-3759,bevacizumab, bortezomib, brigatinib, cabozantinib, capmatinib,ceritinib, cetuximab, CGM097, crizotinib, dabrafenib, dacomitinib,dasatinib, DS-3032b, encorafenib, entrectinib, erdafitinib, erlotinib,everolimus, fam-trastuzumab deruxtecan, figitumumab, gefitinib,gossypol, HDM201, idasanutlin, imatinib, infigratinib, iniparib,lapatinib, larotrectinib, LEE011, lenvatinib, LGX818, lorlatinib,MEK162, MK-8242 SCH 900242, MRTX849, navitoclax, necitumumab, nilotinib,obatoclax, olaparib, OSI-906, osimertinib, palbociclib, panitumumab,PD-0332991, perisofine, pertuzumab, PL225B, repotrectinib, ribociclib,RO5045337, salinomycin, salirasib, SAR405838 MI-77301, sorafenib,sotorasib, sunitinib, tamoxifen, temsirolimus, tipifarnib, tivanitab,tofacitinib, trametinib, trastuzumab, tucatinib, UPR1376, VAL-083,vemurafenib, vemurafenib, vintafolide, or zoptarelin doxorubicin. Insome cases, the RS pathway inhibitor and the targeted therapeutic areadministered together. In some cases, the RS pathway inhibitor and thetargeted therapeutic are administered separately.

In an aspect, there are provided, methods for treating a therapeuticallyresistant cancer in a subject. In some cases, the method comprisesadministering to the subject a therapeutically effective amount of (a) areplication stress (RS) pathway inhibitor, and (b) a targetedtherapeutic. In some cases, the RS pathway inhibitor comprises a RNRinhibitor, an ATR inhibitor, a CHK1 inhibitor, a WEE1 inhibitor, an E2Finhibitor, or a RRM2 inhibitor. In some cases, the RNR inhibitorcomprises Gemcitabine, hydroxyurea, triapine, or5-chloro-2-(n-((1S,2R)-2-(6-fluoro-2,3-dimethylphenyl)-1-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)propyl)sulfamoyl)benzamide.In some cases, the CHK1 inhibitor comprises GDC-0575, prexasertib, orSRA737. In some cases, the therapeutically resistant cancer isecDNA-positive. In some cases, the ecDNA-positive cancer comprises anamplified oncogene on the ecDNA. In some cases, the amplified oncogenecomprises one or more of BRAF, CCND1, CDK4, CDK6, c-Myc, EGFR, ERB2,FGFR, HRAS, IGF1R, KRAS, MDM2, MDM4, MET, MYCL, MYCN, and NRAS. In somecases, the method further comprises administering to the subject atherapeutically effective amount of a targeted therapeutic that inhibitsthe protein product of the amplified oncogene. In some cases, thetargeted therapeutic comprises abemaciclib, ado-trastuzumab emtansine,afatinib, alectinib, ALRN-6924, AMG232, AMG-510, apatinib, ARS-3248,AXL1717, AZD-3759, bevacizumab, bortezomib, brigatinib, cabozantinib,capmatinib, ceritinib, cetuximab, CGM097, crizotinib, dabrafenib,dacomitinib, dasatinib, DS-3032b, encorafenib, entrectinib, erdafitinib,erlotinib, everolimus, fam-trastuzumab deruxtecan, figitumumab,gefitinib, gossypol, HDM201, idasanutlin, imatinib, infigratinib,iniparib, lapatinib, larotrectinib, LEE011, lenvatinib, LGX818,lorlatinib, MEK162, MK-8242 SCH 900242, MRTX849, navitoclax,necitumumab, nilotinib, obatoclax, olaparib, OSI-906, osimertinib,palbociclib, panitumumab, PD-0332991, perisofine, pertuzumab, PL225B,repotrectinib, ribociclib, RO5045337, salinomycin, salirasib, SAR405838MI-77301, sorafenib, sotorasib, sunitinib, tamoxifen, temsirolimus,tipifarnib, tivanitab, tofacitinib, trametinib, trastuzumab, tucatinib,UPR1376, VAL-083, vemurafenib, vemurafenib, vintafolide, or zoptarelindoxorubicin. In some cases, the RS pathway inhibitor and the targetedtherapeutic are administered together. In some cases, the RS pathwayinhibitor and the targeted therapeutic are administered separately.

In an aspect, there are provided compositions comprising a replicationstress (RS) pathway inhibitor and a targeted therapeutic. In some cases,the RS pathway inhibitor comprises a RNR inhibitor, an ATR inhibitor, aCHK1 inhibitor, a WEE1 inhibitor, an E2F inhibitor, or a RRM2 inhibitor.In some cases, the RNR inhibitor comprises Gemcitabine, hydroxyurea,triapine, or5-chloro-2-(n-((1S,2R)-2-(6-fluoro-2,3-dimethylphenyl)-1-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)propyl)sulfamoyl)benzamide.In some cases, the CHK1 inhibitor comprises GDC-0575, prexasertib, orSRA737. In some cases, the targeted therapeutic targets a proteinproduct of an oncogene. In some cases, the oncogene comprises BRAF,CCND1, CDK4, CDK6, c-Myc EGFR. ERB2, FGFR, HRAS, IGF1R, KRAS, MDM2,MDM4, MYCL, MYCN, MET, or NRAS. In some cases, the targeted therapeuticcomprises abemaciclib, ado-trastuzumab emtansine, afatinib, alectinib,ALRN-6924, AMG232, AMG-510, apatinib, ARS-3248, AXL1717, AZD-3759,bevacizumab, bortezomib, brigatinib, cabozantinib, capmatinib,ceritinib, cetuximab, CGM097, crizotinib, dabrafenib, dacomitinib,dasatinib, DS-3032b, encorafenib, entrectinib, erdafitinib, erlotinib,everolimus, fam-trastuzumab deruxtecan, figitumumab, gefitinib,gossypol, HDM201, idasanutlin, imatinib, infigratinib, iniparib,lapatinib, larotrectinib, LEE011, lenvatinib, LGX818, lorlatinib,MEK162, MK-8242 SCH 900242, MRTX849, navitoclax, necitumumab, nilotinib,obatoclax, olaparib, OSI-906, osimertinib, palbociclib, panitumumab,PD-0332991, perisofine, pertuzumab, PL225B, repotrectinib, ribociclib,RO5045337, salinomycin, salirasib, SAR405838 MI-77301, sorafenib,sotorasib, sunitinib, tamoxifen, temsirolimus, tipifarnib, tivanitab,tofacitinib, trametinib, trastuzumab, tucatinib, UPR1376, VAL-083,vemurafenib, vemurafenib, vintafolide, or zoptarelin doxorubicin. Insome cases, the RS pathway inhibitor is a CHK1 inhibitor and thetargeted therapeutic is an EGFR inhibitor. In some cases, thecomposition comprises one or more pharmaceutically acceptableexcipients.

In an aspect, there are provided methods for treating cancer in asubject. In some cases, the method comprises administering to thesubject a therapeutically effective amount of a first targetedtherapeutic until the cancer in the subject develops resistance to thefirst targeted therapeutic, followed by administering to the subject atherapeutically effective amount of a replication stress (RS) pathwayinhibitor, thereby treating the cancer. In some cases, the firsttargeted therapeutic comprises abemaciclib, ado-trastuzumab emtansine,afatinib, alectinib, ALRN-6924, AMG232, AMG-510, apatinib, ARS-3248,AXL1717, AZD-3759, bevacizumab, bortezomib, brigatinib, cabozantinib,capmatinib, ceritinib, cetuximab, CGM097, crizotinib, dabrafenib,dacomitinib, dasatinib, DS-3032b, encorafenib, entrectinib, erdafitinib,erlotinib, everolimus, fam-trastuzumab deruxtecan, figitumumab,gefitinib, gossypol, HDM201, idasanutlin, imatinib, infigratinib,iniparib, lapatinib, larotrectinib, LEE011, lenvatinib, LGX818,lorlatinib, MEK162, MK-8242 SCH 900242, MRTX849, navitoclax,necitumumab, nilotinib, obatoclax, olaparib, OSI-906, osimertinib,palbociclib, panitumumab, PD-0332991, perisofine, pertuzumab, PL225B,repotrectinib, ribociclib, RO5045337, salinomycin, salirasib, SAR405838MI-77301, sorafenib, sotorasib, sunitinib, tamoxifen, temsirolimus,tipifarnib, tivanitab, tofacitinib, trametinib, trastuzumab, tucatinib,UPR1376, VAL-083, vemurafenib, vemurafenib, vintafolide, or zoptarelindoxorubicin. In some cases, the RS pathway inhibitor comprises a RNRinhibitor, an ATR inhibitor, a CHK1 inhibitor, a WEE1 inhibitor, an E2Finhibitor, a RRM1 inhibitor, or a RRM2 inhibitor. In some cases, the RNRinhibitor comprises Gemcitabine, hydroxyurea, triapine, or5-chloro-2-(n-((1S,2R)-2-(6-fluoro-2,3-dimethylphenyl)-1-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)propyl)sulfamoyl)benzamide.In some cases, the CHK1 inhibitor comprises GDC-0575, prexasertib, orSRA737. In some cases, the cancer is determined to be ecDNA-positiveprior to administration of the RS pathway inhibitor. In some cases, theecDNA comprises an amplified oncogene. In some cases, the amplifiedoncogene comprises one or more of BRAF, CCND1, CDK4, CDK6, c-Myc. EGFR,ERB2. FGFR, HRAS, IGF1R, KRAS, MDM2, MDM4, MET, MYCL, MYCN, and NRAS. Insome cases, the method further comprises administering to the subject asecond targeted therapeutic that inhibits the protein product of theamplified oncogene. In some cases, the second targeted therapeuticcomprises abemaciclib, ado-trastuzumab emtansine, afatinib, alectinib,ALRN-6924, AMG232, AMG-510, apatinib, ARS-3248, AXL1717, AZD-3759,bevacizumab, bortezomib, brigatinib, cabozantinib, capmatinib,ceritinib, cetuximab, CGM097, crizotinib, dabrafenib, dacomitinib,dasatinib, DS-3032b, encorafenib, entrectinib, erdafitinib, erlotinib,everolimus, fam-trastuzumab deruxtecan, figitumumab, gefitinib,gossypol. HDM201, idasanutlin, imatinib, infigratinib, iniparib,lapatinib, larotrectinib, LEE011, lenvatinib, LGX818, lorlatinib,MEK162, MK-8242 SCH 900242, MRTX849, navitoclax, necitumumab, nilotinib,obatoclax, olaparib. OSI-906, osimertinib, palbociclib, panitumumab,PD-0332991, perisofine, pertuzumab, PL225B, repotrectinib, ribociclib,RO5045337, salinomycin, salirasib, SAR405838 MI-77301, sorafenib,sotorasib, sunitinib, tamoxifen, temsirolimus, tipifarnib, tivanitab,tofacitinib, trametinib, trastuzumab, tucatinib, UPR1376, VAL-083,vemurafenib, vemurafenib, vintafolide, orzoptarelin doxorubicin. In somecases, the first targeted therapeutic is administered in combinationwith the RS inhibitor, the second targeted therapeutic, or both.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

An understanding of the features and advantages of the present inventionwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of theinvention are utilized, and the accompanying drawings of which:

FIG. 1 shows ecDNA enable cancer cells to resist therapeutic pressure byaltering oncogene dependency and this can be overcome by combinationtreatment with RNR inhibition using gemcitabine and another therapeuticagent.

FIG. 2 shows rapid preclinical resistance (in vitro and in vivo) totreatment with a selective KRAS^(G12C) inhibitor, adagrasib, incolorectal cancer (CRC) models is strongly associated with ecDNA.

FIG. 3 shows ribonucleotide reductase (RNR) & CHK1 function inreplication stress response.

FIG. 4 shows ecDNA-driven tumor cells are more sensitive to inhibitionof RNR or CHK1.

FIG. 5 shows differential sensitivity of ecDNA-positive (ecDNA(+))versus ecDNA-negative (ecDNA(−)) cohort to inhibition of RNR usinggemcitabine in a 3D colony formation assay.

FIG. 6 shows HeLa methotrexate-resistant (MTX-R) ecDNA in a model assay.

FIG. 7 shows CHK1 target validation in HeLa MTX-R model.

FIG. 8 shows ecDNA mediates an important and clinically distinctmechanism of resistance to targeted therapies.

FIG. 9 shows fluorescence in situ hybridization (FISH) images ofparental HeLa cells (left panels) MTX-R HeLa cells (right panel).

FIG. 10 illustrates the study using barcoding and single cell RNAseq forelucidation of pre-existing versus de-novo ecDNA-mediated resistancemechanisms.

FIG. 11 illustrates the study using barcoding with replicate populationsthat develop methotrexate resistance to elucidate pre-existing versusde-novo ecDNA-mediated resistance.

FIG. 12 shows structurally distinct RNR inhibitors induce replicationstress markers in Colo320 system with correlation to ecDNA and relativesensitivity.

FIG. 13 shows ecDNA+ cells are more sensitive to CHK1 inhibitor (CHK1i)than ecDNA− cells.

FIG. 14 shows assays and tumor cell models of ecDNA-dependentsensitivity to CHK1 inhibitors.

FIG. 15 shows growth of KRAS inhibitor (KRASi) resistant tumors in micetreated with various agents.

FIG. 16 shows ecDNA in tumors of mice treated with various agents.

FIG. 17 shows tumor growth in mice treated with various agents.

FIG. 18 shows event free survival in mice treated with various agents.

FIG. 19 shows ecDNA counts in cells from tumors in mice treated withvarious agents.

FIG. 20 shows KRAS ecDNA providing ex vivo propagation of resistanttumor cells.

FIG. 21 shows inhibition of tumor growth in mice by the KRASi,adagrasib, and by the combination of adagrasib and the RNRi,gemcitabine.

FIG. 22 shows inhibition of tumor growth in mice by RNRi, gemcitabine,and KRASi, adagrasib.

FIG. 23 shows inhibition of tumor growth in mice by RNRi, gemcitabine,and KRASi, adagrasib, and CHK1i, prexasertib.

FIG. 24 shows growth of, and ecDNA content in, of SNU16 cells resistantto infigratinib.

FIG. 25 shows growth of FGFR2 ecDNA-driven SNU16 cells in the presenceof various agents.

FIG. 26 shows growth of EGFR ecDNA-driven SNU16 cells in the presence ofvarious agents.

FIG. 27 shows sensitivity of ecDNA+ and ecDNA− cell lines to WEE1inhibition.

FIG. 28 shows adavosertib abrogation of methotrexate resistance inecDNA+ cells.

FIG. 29 shows PD1066285 abrogation of methotrexate resistance in ecDNA+cells.

FIG. 30 shows adavosertib prevents resistance of infigratinib treatmentin SNU16 cells.

FIG. 31 shows sensitivity of ecDNA+ cells to WEE1 inhibition.

FIG. 32 shows WEE1 inhibition prevents methotrexate resistance.

FIG. 33 shows a comparison in sensitivity of HeLa ecDNA+ and HeLa ecDNA−cells to WEE1 knockout.

FIG. 34 shows PARG inhibition delays resistance formation toinfigratinib treatment of SNU16 cells.

FIG. 35 PARG inhibition prevents methotrexate resistance.

FIG. 36 shows sensitivity of ecDNA+ and ecDNA− cell lines to ATRinhibition.

FIG. 37 shows ATR inhibition prevents methotrexate resistance.

FIG. 38 shows replication stress of ecDNA+ cells.

FIG. 39A shows replication fork speed of ecDNA+ and ecDNA− cells.

FIG. 39B shows replication fork speed of ecDNA+ and ecDNA− cells.

FIGS. 40A-40C show inhibition of RNR blocks nucleotide synthesis,enhanced replication stress, and reduced cellular transformation inecDNA+ compared to ecDNA− tumor cells. FIG. 40A shows RNR inhibitionalters cell-proliferation/transformation. FIG. 40B shows FISH imagesquantifying changes in ecDNA carrying amplified oncogene counts. FIG.40C shows inhibition of RNR results in blocked nucleotide synthesis andenhanced replication stress.

FIGS. 41A-41B shows ecDNA+ cells have increased replication stress inresponse to CHK1i. FIG. 41A shows increased sensitivity of ecDNA+ cellscompared with ecDNA-cells to CHK1 inhibition. FIG. 41B showsssDNA-damage induced replication stress with CHK1 pathway inhibition.

FIGS. 42A-42B shows increased replication fork dysfunction in ecDNA+cancer cells compared with ecDNA− cells with RNR pathway inhibition.FIG. 42A shows DNA fiber analysis of ecDNA+ and ecDNA− cells treatedwith RNR inhibitor. FIG. 42B shows replication fork speed in ecDNA+ andecDNA− cells treated with RNR inhibitor.

FIG. 43A-43B shows increased replication fork dysfunction in ecDNA+cells compared with ecDNA− cells with CHK1 inhibition. FIG. 43B showsDNA fiber analysis of ecDNA+ and ecDNA− cells treated with CHK1inhibitor. FIG. 42B shows replication fork speed in ecDNA+ and ecDNA−cells treated with CHK1 inhibitor.

FIG. 44 shows reduced protein levels of RRM2 in cells treated withoncoprotein inhibitors.

FIG. 45 shows inhibition of tumor growth in mice by WEE1i, adavosertib,or PD0166285; KRASi, adagrasib, and the combination.

DETAILED DESCRIPTION

Numerous oncogene-directed therapies have demonstrated clinical efficacyagainst mutated or activated fusion oncogene targets, however these sametherapies do not always yield good objective response rate (ORR) orprogression-free survival (PFS) against tumors, especially when the sameoncogene is amplified. Despite considerable effort, the oncology fieldhas failed to address this significant unmet need cancer populationcharacterized by amplified oncogenes. Data suggests a substantialproportion of these amplifications are focal amplifications that in somecases occur on extrachromosomal DNA (ecDNA), and this ecDNA phenomenonmay account for lack of treatment success.

A SNU16 gastric cancer model shown in FIG. 1 recapitulates the clinicalobservation of “non-responsiveness” to targeted FGFR inhibitor therapyin the case of FGFR2 amplified cancers. The tumor cells predominantlyharbor ecDNA amplified MYC and FGFR2. Although not shown here, the tumorcells demonstrate initial sensitivity to the FGFR inhibitor therapy,with substantial cell reduction in the first two weeks of treatment.However, by five weeks the cell line is growing normally again and iscompletely resistant to FGFR inhibition. This rapid resistance iscorrelated with a reduction in FGFR2 on ecDNA and a striking increase inEGFR on ecDNA. The underlying ecDNA machinery enables rapid oncogenerepertoire change in response to targeted therapeutic pressure. Thekinetics of this evolution in vitro are consistent with a clinical“non-responder” phenotype, despite the fact that the original tumorpopulation was largely sensitive to FGFR inhibition. These findings helpaccount for the observed lack of clinical efficacy demonstrated foroncogene-directed therapies (e.g. FGFR, EGFR. MET) when these oncogenesare activated via amplification. Therapeutically targeting theunderlying ecDNA machinery constitutes an unprecedented and orthogonalstrategy to overcome therapeutic resistance associated with oncogeneamplified tumors. In accordance, FIG. 25 shows that simultaneousinhibition of FGFR2 with infigratinib and ecDNA with RNR inhibitorgemcitabine completely inhibits cell growth and prevents development ofresistance. Similarly. FIG. 26 shows that cells that become resistant toinfigratinib become mainly driven by EGFR amplification on ecDNA and areinitially sensitive to EGFR inhibitor erlotinib but develop secondaryresistance within three weeks. However, simultaneous inhibition of EGFRby erlotinib and RNR inhibition by gemcitabine completely inhibits cellgrowth and prevents development of secondary resistance. Importantly,upfront simultaneous inhibition of FGFR2 and EGFR by infigratinib anderlotinib, respectively, simply delays development of resistance.

A second in vivo model shown in FIG. 2 exemplifies another resistancemechanism associated with ecDNA wherein targeted therapy againstactivating mutant KRAS^(G12C) using the selective KRASG12C inhibitorMRTX849 results in initial tumor reduction followed by rapid resistanceand regrowth. The initial tumor does not have significant ecDNA, whereasthe resistant tumor shows clear evidence of ecDNA harboring amplifiedKRAS^(G12C). Prior published data is consistent with a similar likelyecDNA-mediated phenomenon of oncogene amplification driving resistanceto EGFR and BRAF inhibitors in BRAF mutant colorectal cancer. Theseresults indicate a unique utility for an ecDNA-directed therapy toaddress a major resistance mechanism associated with mutant activatedMAPK pathway inhibition.

The oncology field has struggled to find the appropriate geneticbackground/sensitivity signature to successfully deploy ReplicationStress (RS)-targeted therapies including ATR. CHK1 and WEE1. ATRinhibitors are showing some potential in ATM-mutant prostate cancer, butstudies are ongoing. Synthetic lethality associated with oncogeneamplification has been proposed (such as MYC, MYCN, MYCL, CCNE1 inparticular, as they have been associated with increased RS), along withother genetic alterations and/or HPV+. The data supporting thesedependencies were far from conclusive and too heterogeneous. Providedherein are methods wherein ecDNA-directed inhibition (inhibition of areplication stress pathway component) exhibits synthetic lethality witha cancer-targeted agent. In some cases, synthetic lethality withRS-targeted agents includes synthetic lethality of a cancer targetedagent with inhibition of a replication stress pathway component, such aswith ribonucleotide reductase (RNR) or CHK1 inhibitors. In some cases, atumor background is identified as hyper-sensitive to a replicationstress pathway inhibition agent and allows a sufficient therapeuticindex to enable tolerated doses that are efficacious. In some cases,inhibition of a component of the replication stress pathway results inreduced ecDNA copy number and enhanced cytotoxicity in ecDNA positivecells. In some cases, enhanced cytotoxicity results from the combinationof the inhibition of a component of the replication stress pathway andinhibition of a cancer-target, such as an oncogene.

Methods of Treatment

In an aspect, provided herein are methods of treating cancer in asubject, for example methods of treating a tumor or tumor cells in asubject. In some cases, methods herein comprise administering areplication stress pathway agent (RSPA) in an amount sufficient toinduce replication stress in the tumor or tumor cells. In some cases,the method further comprises administering a cancer-targeted therapeuticagent. In some cases, the tumor or tumor cells have an extrachromosomaldeoxynucleic acid (ecDNA) signature. In some cases, growth or size ofthe tumor or growth or number of tumor cells is reduced.

In an aspect of methods herein, a tumor or tumor cells are determined tohave an ecDNA signature. In some cases, a tumor or tumor cells aredetermined to have an ecDNA signature when the tumor or tumor cells haveone or more characteristics associated with ecDNA+ tumors or tumorcells. For example, in some cases, the ecDNA signature is selected fromthe group consisting of a gene amplification; a p53 loss of functionmutation; absence of microsatellite instability (MSI-H); a low level ofPD-L1 expression; a low level of tumor inflammation signature (TIS); alow level of tumor mutational burden (TMB); an increased frequency ofallele substitutions, insertions, or deletions (indels); and anycombination thereof.

In an aspect of methods herein, the method further comprisesadministering a cancer-targeted therapeutic agent, directed to anactivity of a protein product of a target gene. In some cases, thetreatment with the cancer-targeted therapeutic agent and the RSPAreduces amplification or expression of the target gene in the tumor ortumor cells. In some cases, the cancer-targeted therapeutic agent isadministered prior to the RSPA. In some cases, the cancer-targetedtherapeutic agent is administered concurrently with the RSPA. In somecases, the cancer-targeted therapeutic agent is administered prior tothe RSPA.

In an aspect of methods herein, the tumor or tumor cells have an ecDNAsignature. In some cases, the tumor or tumor cells develop the ecDNAsignature after administration of the cancer-targeted therapeutic agent.In some cases, the tumor or tumor cells develop the ecDNA signatureprior to treatment. In some cases, the method prevents an increase ofecDNA in the tumor or tumor cells.

In an aspect of methods provided herein, the cancer-targeted therapeuticagent targets a protein product of an oncogene. In some cases, theoncogene comprises a point mutation, an insertion, a deletion, a fusion,or a combination thereof. In some cases, the cancer-targeted therapeuticagent targets a gene selected from the group consisting of AKT, ALK, AR,BCL-2, BCR-ABL, BRAF, CDK4, CDK6, c-MET, EGFR, ER. ERBB3, ERRB2, FAK,FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GR, HRAS, IGF1R, KIT, KRAS, MCL-1,MDM2, MDM4, MTOR. MYC, MYCL, MYCN, NRAS, NRG1, NTRK1, NTRK2, NTRK3,PDGFR, PIK3CA/B, PIK3Cδ. RET, and ROS1. In some cases, thecancer-targeted therapeutic agent targets one or more genes provided inTable 1.

In an aspect of methods provided herein, the tumor or tumor cellscomprise an amplification of a first gene or portion thereof. In somecases, the first gene is an oncogene. In some cases, the first gene is adrug resistance gene. In some cases, the amplification is present onecDNA. In some cases, the first gene is selected from the groupconsisting of AKT, ALK, AR, BCL-2, BCR-ABL, BRAF, CDK4, CDK6, c-MET,EGFR, ER, ERRB2, ERBB3, FAK, FGFR1. FGFR2, FGFR3, FGFR4, FLT3, GR, HRAS,IGF1R, KRAS, KIT, MCL-1, MDM2, MDM4MTOR, NRAS, PDGFR, RET, and ROS1. Insome cases, the first gene comprises one or more genes provided inTable 1. In some cases, the cancer-targeted therapeutic agent isdirected against the first gene. In some cases, the subject has not beenpreviously treated with the cancer-targeted therapeutic agent. In somecases, the tumor or tumor cells have not been previously treated withthe cancer-targeted therapeutic agent. In some cases, the methodprevents an increase of ecDNA in the tumor or tumor cells. In somecases, the method prevents a further increase in the amplification ofthe first gene. In some cases, such further amplification occurs if onlythe cancer-targeted therapeutic agent is administered, but when thetreatment includes both the cancer-targeted therapeutic agent and theRSPA, the further increase in amplification is inhibited or prevented.

In an aspect of methods provided herein, the tumor or tumor cells areresistant or non-responsive to a previous therapeutic agent prior totreatment with the cancer-targeted therapeutic agent and the RSPA. Insome cases, the tumor or tumor cells have been previously treated withthe previous therapeutic agent. In some cases, the subject has beenpreviously treated with the previous therapeutic agent. In some cases,after a period of treatment with the previous therapeutic agent, thetumor or tumor cells become resistant or non-responsive to such previousagent, and with the methods herein, when such tumor or tumor cells aretreated with the cancer-targeted therapeutic agent (an agent that is, insome instances, different from the previous therapeutic agent) and theRSPA, the growth of the tumor or tumor cells is inhibited. In somecases, the treatment reduces the amount or level of ecDNA in the treatedtumor or tumor cells or prevents a further increase in ecDNA amount orlevel.

In an aspect of methods provided herein, the cancer-targeted therapeuticagent is directed to an activity of a protein product of a target gene.In some cases, treatment with the cancer-targeted therapeutic agent andthe RSPA reduces amplification or expression of the target gene in thetumor or tumor cells. In some cases, the target gene is an oncogene, adrug-resistance gene, a therapeutic target gene, or a checkpointinhibitor gene. In some cases, the target gene is selected from thegroup consisting of KRAS, HRAS, NRAS, BRAF, EGFR, FGFR1, FGFR2, FGFR3,FGFR4, ALK, ROS1, RET, PDGFR, c-MET, IGF1R, FAK, BCR-ABL, MCL-1, CDK4,CDK6, ERRB2, ERBB3, MDM2, MTOR, FLT3, KIT, AKT, BCL-2, AR, ER, GR andMDM4. In some cases, the target gene comprises one or more genesprovided in Table 1. In some cases, the target gene is found on or foundamplified on ecDNA and treatment with the cancer-targeted therapeuticagent and the RSPA reduces ecDNA, including ecDNA comprising copies ofthe target gene.

In various aspects of methods provided herein, in some cases, the ecDNAsignature of the tumor or tumor cells is known prior to beginningtreatment of the tumor or tumor cells. For example, the tumor or tumorcells are biopsied or otherwise collected and assayed for one or moreecDNA signatures. In some cases, a determination of how to treat thetumor or tumor cells is based, in whole or in part, the presence orabsence of an ecDNA signature. In some cases, the ecDNA signature isknown after treatment of the tumor or tumor cells has commenced.

In an aspect of methods provided herein, the method of treatment with acancer-targeted therapeutic agent and an RSPA improves an objectiveresponse rate and/or extends a duration of treatment response ascompared to treatment with the cancer-targeted therapeutic agent in theabsence of the RSPA. In some cases, the method increases a period ofprogression free survival as compared to treatment with thecancer-targeted therapeutic agent in the absence of the RSPA.

In an aspect, there are provided methods of treating an ecDNA-associatedtumor or tumor cells. In some cases, the method comprises, administeringa RSPA and a cancer-targeted therapeutic agent to a subject identifiedas having a tumor or tumor cells having ecDNA. In some cases, growth orsize of the tumor or growth or number of the tumor cells is decreased asa result of treatment.

In an aspect of methods provided herein, the tumor or tumor cells of thesubject are identified as having an ecDNA signature. In some cases, theecDNA signature is selected from the group consisting of a geneamplification; a p53 loss of function mutation; absence ofmicrosatellite instability (MSI-H); a low level of PD-L1 expression; alow level of tumor inflammation signature (TIS); a low level of tumormutational burden (TMB); an increased frequency of allele substitutions,insertions, or deletions (indels); and any combination thereof.

In an aspect of methods provided herein, the tumor or tumor cells areidentified as having ecDNA by imaging ecDNA in cells, detecting ecDNAusing an oncogene binding agent, or by DNA sequencing. In some cases,ecDNA is identified in circulating tumor DNA.

In various aspects of methods provided herein, the tumor or tumor cellsare comprised by a solid tumor. In some cases, presence of ecDNA in thesolid tumor is reduced or abolished as a result of treatment with acancer-targeted therapeutic agent and an RSPA. In some cases, a level ofecDNA is reduced in the solid tumor after treatment as compared to thelevel of ecDNA prior to treatment. In some cases, a level of oncogeneamplification and/or a level of copy number variation (CNV) in the solidtumor is reduced after treatment with a cancer-targeted therapeuticagent and an RSPA as compared to the level of oncogene amplificationand/or CNV in the solid tumor prior to treatment.

In various aspects of methods provided herein, the tumor or tumor cellsinclude circulating tumor cells. In some cases, presence of ecDNA in thecirculating tumor cells is reduced or abolished as a result of treatmentwith a cancer-targeted therapeutic agent and an RSPA. In some cases, alevel of ecDNA is reduced in the circulating tumor cells after treatmentas compared to the level of ecDNA prior to treatment. In some cases, alevel of oncogene amplification and/or a level of copy number variation(CNV) in the circulating tumor cells is reduced after treatment ascompared to the level of oncogene amplification and/or CNV in thecirculating tumor cells prior to treatment. In some cases, the presenceor level of ecDNA is identified in circulating tumor DNA.

In various aspects of methods provided herein that employ treatment witha RSPA and a cancer-targeted therapeutic agent, the RSPA is selectedfrom the group consisting of a RNR inhibitor, an ATR inhibitor, a CHK1inhibitor, a WEE1 inhibitor, and a PARG inhibitor. In some cases, theRNR inhibitor is selected from the group consisting of gemcitabine,hydroxyurea, triapine,5-chloro-2-(n-((1S,2R)-2-(6-fluoro-2,3-dimethylphenyl)-1-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)propyl)sulfamoyl)benzamide,clofarabine, fludarabine, motexafin gadolinium, cladribine,tezacitabine, and COH29(N-[4-(3,4-dihydroxyphenyl)-5-phenyl-1,3-thiazol-2-yl]-3,4-dihydroxybenzamide).In some cases, the CHK1 inhibitor is selected from the group consistingof GDC-0575, prexasertib, LY-2880070, SRA737, XCCS-605B, rabusertib(LY-2603618), SCH-900776, RG-7602, AZD-7762, PF-477736, and BEBT-260. Insome cases, the WEE1 inhibitor is selected from the group consisting ofAZD1775 (MK1775), ZN-c3, Debio 0123, IMP7068, SDR-7995, SDR-7778,NUV-569, PD0166285, PD0407824, SC-0191, DC-859/A, bosutinib, and Bos-I.In some cases, the ATR inhibitor is selected from the group consistingof RP-3500, M-6620, berzosertib (M-6620, VX-970; VE-822), AZZ-6738,AZ-20, M-4344 (VX-803), BAY-1895344, M-1774, IMP-9064, nLs-BG-129,SC-0245, BKT-300, ART-0380, ATRN-119, ATRN-212, NU-6027.

In various aspects of methods provided herein that employ treatment witha cancer-targeted therapeutic agent and an RSPA, the cancer targetedtherapeutic agent is selected from the group consisting of abemaciclib,ado-trastuzumab emtansine, afatinib, alectinib, ALRN-6924, AMG232,AMG-510, apatinib, ARS-3248, AXL1717, AZD-3759, bevacizumab, bortezomib,brigatinib, cabozantinib, capmatinib, ceritinib, cetuximab, CGM097,crizotinib, dabrafenib, dacomitinib, dasatinib, DS-3032b, encorafenib,entrectinib, erdafitinib, erlotinib, everolimus, fam-trastuzumabderuxtecan, figitumumab, gefitinib, gossypol, HDM201, idasanutlin,imatinib, infigratinib, iniparib, lapatinib, larotrectinib, LEE011,lenvatinib. LGX818, lorlatinib, MEK162, MK-8242 SCH 900242, MRTX849,navitoclax, necitumumab, nilotinib, obatoclax, olaparib, OSI-906,osimertinib, palbociclib, panitumumab. PD-0332991, perisofine,pertuzumab, PL225B, repotrectinib, ribociclib, RO5045337, salinomycin,salirasib, SAR405838 MI-77301, sorafenib, sotorasib, sunitinib,tamoxifen, temsirolimus, tipifarnib, tivanitab, tofacitinib, trametinib,trastuzumab, tucatinib, UPR1376, VAL-083, vemurafenib, vemurafenib,vintafolide, and zoptarelin doxorubicin. In some cases, the cancertargeted therapeutic agent targets a protein encoded by one or moregenes provided in Table 1.

TABLE 1 Example Genes Gene Description ABCB(1-11) ABC binding cassettesubfamily A ABCC(1-13) ABC binding cassette subfamily C ABCG(1-5) ABCbinding cassette subfamily G ABL1 encodes ABL kinase AKT(1-3) familyencoding AKT serine/threonine kinases ALK anaplastic lymphoma kinaseNRC3C4 (AR) androgen receptor BCL2 encodes BCL-2 apoptosis regulator BCRencodes breakpoint cluster region protein BRAF encodes B-rafserine/threonine kinase CCND1 Cyclin D1 CCNE1 Cyclin E1 CDK4 celldivision protein kinase 4 CDK6 cell division protein kinase 6 EGFREpidermal growth factor receptor (also known as ERBBI and HER1)ERBB(1-4) HER1-4 family of receptor proteins, including EGFR ESR(1-2)estrogen receptor (alpha and beta) FAK focal adhesion kinase FGFR(1-4)FGFR1-4 family of receptor proteins FLT3 FMS like tyrosine kinase 3, akaCD135 NR3C1 (GR) glucocorticoid receptor HRAS encodes RASGTPase/signaling protein HRAS IGF1R insulin like growth factor receptor(IGF-1R) KIT encodes c-Kit, aka CD117 KRAS encodes RAS GTPase/signalingprotein KRAS MCL1 encodes MCL-1, myeloid leukemia cell differentiationprotein MDM2 mouse double minute 2 MDM4 mouse double minute 4 METencodes c-Met protein (aka HGFR) MTOR encodes mechanistic target ofrapamycin (mTOR) MYC encodes c-Myc MYCL encodes l-Myc MYCN encodes n-MycNRAS encodes RAS GTPase/signaling protein NRAS NRG1 encodes neuregulin 1NTRK(1-3) neurotrophic tyrosine receptor kinase PDGFR encodes plateletderived growth factor receptor NR3C3 (PGR) progesterone receptorPIK3CA/B/D/G encodes phosphatidylinositol 3-kinase subunitsalpha/beta/delta/gamma PIK3Cδ encodes phosphatidylinositol 3-kinasedelta RET encodes RET proto-oncogene ROS1 encodes ROS proto-oncogene 1S100A8(MRP8) encodes S100 calcium binding protein A8

In an aspect of methods provided herein, the RSPA is an RNR inhibitorand the RSPA is administered at a sub-therapeutic dose relative to itsrecommended use as a single agent. In some cases, the RNR inhibitor isgemcitabine. Alternatively, the RNR inhibitor is not gemcitabine orhydroxy urea.

In an aspect of methods provided herein, the RSPA is not gemcitabine. Insome cases, the RSPA is not gemcitabine when the cancer-targetedtherapeutic agent is an EGFR inhibitor.

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Certain Definitions

As used herein the term “about” or “approximately” means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which can depend in part on how the value ismeasured or determined. i.e., the limitations of the measurement system.For example, “about” can mean within 1 or more than 1 standarddeviation, per the practice in the art. As another example, “about” canmean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a givenvalue. With respect to biological systems or processes, the term “about”can mean within an order of magnitude, such as within 5-fold or within2-fold of a value. Where particular values are described in theapplication and claims, unless otherwise stated, the term “about” meanswithin an acceptable error range for the particular value.

The term “subject,” as used herein, generally refers to a vertebrate,such as a mammal (e.g., a human). Mammals include, but are not limitedto, murines, simians, humans, farm animals, sport animals, and pets(e.g., a dog or a cat). Tissues, cells, and their progeny of abiological entity obtained in vivo or cultured in vitro are alsoencompassed. In some embodiments, the subject is a patient. In someembodiments, the subject is symptomatic with respect to a disease (e.g.,cancer). Alternatively, in some cases, the subject is asymptomatic withrespect to the disease. In some cases, the subject does not have thedisease.

The term “biological sample,” as used herein, generally refers to asample derived from or obtained from a subject, such as a mammal (e.g.,a human). Biological samples are contemplated to include but are notlimited to, hair, fingernails, skin, sweat, tears, ocular fluids, nasalswab or nasopharyngeal wash, sputum, throat swab, saliva, mucus, blood,serum, plasma, placental fluid, amniotic fluid, cord blood, emphaticfluids, cavity fluids, earwax, oil, glandular secretions, bile, lymph,pus, microbiota, meconium, breast milk, bone marrow, bone, CNS tissue,cerebrospinal fluid, adipose tissue, synovial fluid, stool, gastricfluid, urine, semen, vaginal secretions, stomach, small intestine, largeintestine, rectum, pancreas, liver, kidney, bladder, lung, and othertissues and fluids derived from or obtained from a subject.

The term “treating” as used herein, generally refers to administering anagent, or carrying out a procedure, for the purposes of obtaining aneffect. In some cases, the effect is prophylactic in terms of completelyor partially preventing a disease or symptom thereof and/or istherapeutic in terms of effecting a partial or complete cure for adisease and/or one or more symptoms of the disease. “Treatment,” as usedherein, may include treatment of a tumor in a mammal, particularly in ahuman, and includes: (a) preventing the disease or a symptom of adisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it (e.g., includingdiseases that may be associated with or caused by a primary disease; (b)inhibiting the disease, i.e., arresting its development; and (c)relieving the disease, i.e., causing regression of the disease. Treatingmay refer to any indicia of success in the treatment or amelioration orprevention of an cancer, including any objective or subjective parametersuch as abatement; remission; diminishing of symptoms or making thedisease condition more tolerable to the patient; slowing in the rate ofdegeneration or decline; or making the final point of degeneration lessdebilitating. The treatment or amelioration of symptoms is based on oneor more objective or subjective parameters; including the results of anexamination by a physician. Accordingly, the term “treating” includesthe administration of the compounds or agents of the present inventionto prevent or delay, to alleviate, or to arrest or inhibit developmentof the symptoms or conditions associated with cancer or other diseases.The term “therapeutic effect” refers to the reduction, elimination, orprevention of the disease, symptoms of the disease, or side effects ofthe disease in the subject.

The term “tumor” or “tumor cells” as used herein, generally refers tocells that grow and divide more than they should or do not die when theyshould. In some cases, tumor cells are present in a solid mass, such asa solid tumor, or in some cases, tumor cells are found in a non-solidform, such as in blood cancers. Tumor or tumor cells also can includemetastasis or metastasizing cells, where cancer cells break away fromthe original (primary) tumor and may form a new tumor in other organs ortissues of the body.

The term “oncogene” as used herein, generally refers to a gene that hasthe potential to cause cancer when inappropriately activated. In tumorsor tumor cells, these genes are often mutated to remove negativeregulatory domains or expressed at high levels.

The term “ecDNA signature” as used herein, generally refers to one ormore characteristics common to tumors or tumor cells that are ecDNA+. Insome cases, the ecDNA signature is selected from the group consisting ofa gene amplification; a p53 loss of function mutation; absence ofmicrosatellite instability (MSI-H); a low level of PD-L1 expression; alow level of tumor inflammation signature (TIS); a low level of tumormutational burden (TMB); an increased frequency of allele substitutions,insertions, or deletions (indels); and any combination thereof. In somecases, ecDNA signature includes a detection or identification of ecDNAusing an imaging technology. In some cases, ecDNA signature does notinclude any imaging or direct detection of ecDNA.

The terms “replication stress pathway agent,” “RSPA,” “replicationstress pathway inhibitor,” and “RS pathway inhibitor” as used herein,generally refer to an agent that causes replication stress in a cell,such as a tumor cell. In some cases, the RSPA is an inhibitor of areplication stress pathway component, where inhibition increasesreplication stress. Replication stress as used herein refers to a stressthat affects DNA replication and/or DNA synthesis and can include but isnot limited to the slowing or stalling of replication fork progressionand/or interference with DNA synthesis. Exemplary replication stresspathway agents include but are not limited to agents that inhibit RNR(ribonucleotide reductase), CHK1 (checkpoint kinase 1). ATR(Rad3-related protein), WEE1, E2F, PARG (poly(ADPribose)glycohydrolase), or RRM2 (ribonucleotide reductase regulatorysubunit 2).

Whenever the term “at least,” “greater than,” or “greater than or equalto” precedes the first numerical value in a series of two or morenumerical values, the term “at least,” “greater than” or “greater thanor equal to” applies to each of the numerical values in that series ofnumerical values. For example, greater than or equal to 1, 2, or 3 isequivalent to greater than or equal to 1, greater than or equal to 2, orgreater than or equal to 3.

Whenever the term “no more than.” “less than,” or “less than or equalto” precedes the first numerical value in a series of two or morenumerical values, the term “no more than,” “less than,” or “less than orequal to” applies to each of the numerical values in that series ofnumerical values. For example, less than or equal to 3, 2, or 1 isequivalent to less than or equal to 3, less than or equal to 2, or lessthan or equal to 1.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the present disclosure and are not meant to limit thedisclosure herein in any fashion. The present examples, along with themethods described herein are presently representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the disclosure herein. Changes therein and other uses which areencompassed within the spirit of the disclosure as defined by the scopeof the claims will occur to those having ordinary skill in the art.

Example 1: Inhibition of RS Pathway in ecDNA-Positive Cancer

FIG. 3 illustrates the replication stress pathway that is activated insome cases by oncogenes such as HPV7 (E7), dNTP depletion, and otherinstances.

CHK1 and RNR function in the replication stress (RS) response pathwaywithin the DNA damage response network. A range of factors in tumorcells may activate the RS pathway to maintain proliferation and survivalduring replication stress. Inhibition of targets in this pathway couldbe synthetically lethal in these tumor cells by elevating the level ofRS to toxic levels. Both RNR and CHK1 are essential; therefore, tworelated challenges with clinical development of RNR and CHK1 inhibitorsis patient selection and therapeutic index.

FIG. 12 illustrates ecDNA-driven tumor cells are more sensitive toinhibition of RNR or CHK1. Multiple structurally distinct inhibitors ofRNR and CHK1 demonstrate ˜5-10 fold enhanced toxicity in ecDNA-driventumor cells compared to matched non-ecDNA bearing cells. Treatment withan RNR inhibitor results in reduced copies of MYC-encoding ecDNA.

Colo320 ecDNA+ (cell line COLO320 DM) and Colo320 ecDNA− (chromosomallyamplified, cell line COLO320 HSR) cells (colorectal adenocarcinoma celllines, ATCC) were treated with three structurally distinct RNRinhibitors, gemcitabine, hydroxyurea (HU), or compound 1 (comp-1(5-chloro-2-(n-((1S,2R)-2-(6-fluoro-2,3-dimethylphenyl)-1-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)propyl)sulfamoyl)benzamide)).As shown in FIG. 12 , ecDNA positive cells (Colo320 DM) were 5-10 foldmore sensitive to the RNR inhibitor as compared to the ecDNA negativecell line (Colo320 HSR). All RNR inhibitors induced the replicationstress marker, phosphorylation at serine positions 317 and 345 of CHK1,and in general the ecDNA positive cells had increased induction of thisreplication stress marker as compared to the ecDNA negative cells. TheRNR inhibitor hydroxyurea (HU) treated ecDNA+ (Colo320 DM) cells andcontrol cells (vehicle treated) were assessed for ecDNA. The vehicletreated cell line had high levels of ecDNA, as expected, whereas aftertreatment with the RNR inhibitor, hydroxyurea, the ecDNA copy number wasmarkedly reduced.

The sensitivity of an ecDNA+ cell line (Colo320 DM) compared with anecDNA− cell line (Colo320 HSR) to four structurally distinct CHK1inhibitors was determined. The two cell lines were treated with GDC0575,Prexasertib, Rabusertib, or SRA737 for seven days. As shown in FIG. 13 ,Colo320 DM ecDNA+ cells displayed about 5-10 fold enhanced sensitivityto CHK1 inhibition compared with Colo320 HSR ecDNA− cells. In contrast,neither inhibiting CHK2 with CCT241533 nor inhibiting DDR target, ATM,with AZD0156 revealed any differential sensitivity between the twolines.

FIG. 5 illustrates differential sensitivity of ecDNA-positive vs.ecDNA-negative cancer cells to inhibition of RNR using gemcitabine in a3-dimensional colony formation assay conducted in soft-agar. A panel ofecDNA+ and ecDNA− cell lines were interrogated in a two-week soft-agardrug sensitivity assay. All lines were treated with a five-point doserange of gemcitabine for two weeks and stained with crystal violet (FIG.5 , top panel) to quantify individual colony counts by ImageJ analysis(plotted in FIG. 5 , bottom panel). All ecDNA+ cell lines (SNU16,Colo320 DM, H716, and H2170) displayed 5-10 fold enhanced sensitivity togemcitabine when compared with ecDNA− cell lines (DLD1, Colo320 HSR,Lovo, and SW48).

FIG. 31 illustrates sensitivity of ecDNA+ cells to inhibition of WEE1using adavosertib in a 3-dimensional colony formation assay conducted insoft-agar. A panel of ecDNA+ cell lines were interrogated in a two-weeksoft agar drug sensitivity assay. All lines were treated with afive-point dose range of adavosertib for two weeks and stained withcrystal violet to quantify individual colony counts. All cell linesdemonstrated sensitivity to adavosertib.

FIG. 32 illustrates abrogation of resistance formation to methotrexateby WEE1 inhibition via adavosertib (MK-1775) in HeLa cells via colonyformation assay. In FIG. 32 left panel, 100 cells were plated withoutmethotrexate for 10 days then treated with DMSO, prexasertib (IC50, 1/3IC50, or 1/9 IC50), or adavosertib (MK-1775) (IC50, 1/3 IC50, or 1/9IC50). In FIG. 32 , right panel, 10000 cells were plated withmethotrexate for 21 days, and simultaneously treated with DMSO,prexasertib (IC50, 1/3 IC50, or 1/9 IC50), or adavosertib(MK-1775)(IC50, 1/3 IC50, or 1/9 IC50).

Sensitivity of an ecDNA+ (Colo320 DM) and an ecDNA− (Colo320 HSR) cellline models was determined by treating each with increasingconcentrations of Prexasertib (CHK1i) over a seven day period. Cellproliferation was determined using MTS assay (FIG. 14 , top left panel),while cytotoxicity was determined by measuring dead-cell proteaseactivity using CytoTox-Glo assay (FIG. 14 , top right panel). ecDNAbearing Colo320 DM showed enhanced sensitivity by 200 fold and 13.71fold to the treatment with CHK1i when analyzing proliferation andcytotoxicity changes compared with Colo320 HSR ecDNA− cells.

The sensitivity of parental CT26WT E3 cells (ecDNA− cell line) andadagrasib-resistant CT26WT E3 cells (ecDNA+) in the presence and absenceof 1 μM adagrasib, to increasing concentrations of Prexasertib (CHK1i)for five days was determined using Cell Titer-glo assay. As shown inFIG. 14 bottom panel, adagrasib-resistant CT26WT E3 ecDNA+ cellsdisplayed about 10 fold enhanced sensitivity to CHK1 inhibition comparedwith non-ecDNA bearing parental CT26WT E3 cells.

HeLa cells were treated with CHK1 inhibitor 10 nM GDC-0575 alone, with100 nM methotrexate alone, or with a combination of the 10 nM GDC-0575and 100 nM methotrexate over the course of three weeks. Cell confluencewas measured via high-content microscopy over the time course. GDC-0575had no effect on cell growth. Methotrexate initially resulted in littleto no cell growth, but after two weeks in culture, the cells began todevelop resistance and cell growth resumed. In contrast, the combinationwith the CHK1 inhibitor prevented development of methotrexateresistance. This effect lasted until termination of the study at eightweeks. FIG. 6 shows data related to ecDNA-positivemethotrexate-resistant HeLa cancer cells. An RNR inhibitor abrogatesrapid ecDNA-mediated resistance to methotrexate treatment, resulting inobserved synthetic lethality. FIG. 9 shows FISH images of parental HeLacells (left panels) and methotrexate-resistant HeLa cells (right panel).This data shows that the untreated HeLa cells have very fewecDNA-positive cells. In contrast, the methotrexate-resistant HeLa cellshave increased levels of ecDNA. Preliminary CHK1 target validation inmethotrexate-resistant HeLa cells is shown in FIG. 7 . This data showslive cell microscopy suggests potential synthetic lethality with CHK1inhibition in the formation of methotrexate resistance, via DHFRamplification, on ecDNA in HeLa cells.

Sensitivity of an ecDNA+ (Colo320 ecDNA+) and an ecDNA− (Colo320 ecDNA−)cell line models to WEE1 inhibition was determined by treating each withincreasing concentrations of either adavosertib or PD0166285 over a 7day period. Cell proliferation was determined using a MTS assay (FIG. 27, top panels), while viability was determined using CellTiter-glo assay(FIG. 27 bottom panels). Colo320 ecDNA+ cells compared with Colo320ecDNA− cells showed enhanced sensitivity to the treatment withadavosertib and PD0166285 when analyzing proliferation and viabilitychanges.

Sensitivity of an ecDNA+ (Colo320 ecDNA+) and an ecDNA− (Colo320 ecDNA−)cell line models to ATR inhibition was determined by treating each withincreasing concentrations of either AZD6738 or BAY1895344 over a 7 dayperiod. Cell proliferation was determined using a MTS assay (FIG. 36 ,top panels), while viability was determined using CellTiter-glo assay(FIG. 36 , bottom panels). Colo320 ecDNA+ cells compared with Colo320ecDNA− cells showed enhanced sensitivity to the treatment with AZD6738and BAY1895344 when analyzing proliferation and viability changescompared.

HeLa cells were treated with a WEE1 inhibitor, 0.01-0.3 μM adavosertibalone, with 1 nM-100 nM PD0166285, with 100 nM methotrexate alone, orwith a combination of 0.01-0.3 μM adavosertib and 100 nM methotrexate ora combination of 1 nM-100 nM PD0166285 and 100 nM methotrexate over thecourse of three weeks. Cell confluence was measured via high-contentmicroscopy over the time course. Methotrexate initially resulted inlittle to no cell growth, but after two weeks in culture, the cellsbegan to develop resistance and cell growth resumed. In contrast, thecombination with adavosertib prevented development of methotrexateresistance. FIG. 28 shows data related to adavosertib (MK1775)abrogation of methotrexate resistance in ecDNA-positive methotrexateresistant HeLa cancer cells as measured by NucRed. FIG. 29 shows datarelated to PD0166285 abrogation of methotrexate resistance inecDNA-positive methotrexate resistant HeLa cancer cells.

HeLa cells were treated with a PARG inhibitor, 3-100 μM PD00017273 with(FIG. 35 top panel) or without (FIG. 35 bottom panel) 100 nMmethotrexate over the course of 3 weeks. Cell confluence was measuredvia high-content microscopy over the time course. Methotrexate initiallyresulted in little to no cell growth, but after two weeks in culture,the cells began to develop resistance and cell growth resumed. Incontrast, the combination with PD00017273 prevented development ofmethotrexate resistance.

HeLa cells were treated with ATR inhibitors 300 nM-10 μM AZD6738 or10-300 nM BAY1895344 with (FIG. 37 , top panels) or without (FIG. 37 ,bottom panels) 100 nM methotrexate over the course of 3 weeks. Cellconfluence was measured via high-content microscopy over the timecourse. AZD6738 showed synthetic lethality with 100 nM methotrexate buton its own, AZD6738 did not affect HeLa cell growth. However. BAY1895344showed toxicity on its own above 10 nM.

ecDNA mediates an important and clinically distinct mechanism ofresistance to targeted therapies. A model for this is shown in FIG. 8 .This data suggests that there are immediate opportunities for utility ofecDNA-directed therapies, such as the use of one more RS-pathwaytargeting agents, including but not limited to those targeting RNR, ATR,CHK1 and WEE1, as a single agent or in combination with other therapies.First, tumors with non-mutant amplified oncogenes for which there are noapproved targeted therapies (e.g. FGFR, EGFR, MET, KRAS, MDM2amplifications). Second, tumors treated with one or more targeted agentswhere acquired resistance of the cancer develops when using the one ormore targeted agents that directly inhibit activating mutant forms ofcertain oncoproteins (e.g. KRAS. BRAF, EGFR) as a consequence of focalamplification such as ecDNA-based amplification of the target geneitself.

Example 2: Routes to Resistance in ecDNA-Positive Cancer

In order to understand the mechanism of emergence of resistance totherapy mediated by ecDNA, it was determined whether resistant clonesharboring ecDNA were pre-existing or if they were formed de-novo undertherapy pressure. Barcoding experiments were performed in HeLa cellsthat become resistant to prolonged treatment with DHFR inhibitormethotrexate (MTX) through generation of ecDNA that harbors DHFR, henceovercoming MTX pressure.

Barcoding in Combination with Single Cells RNAseq Analysis.

The initial naïve population of cells was barcoded by stable lentiviralmediated integration of a barcode sequence into the genome of each cell.This barcode will also be expressed in the RNA of each cell. Single cellRNAseq analysis of cells will identify cells (though barcode) thatharbor high expression of DHFR, indicative of presence of extra DH FRcopies on ecDNA. Following several weeks of MTX pressure and generationof resistant cells, single cell RNAseq was performed again to identifythe cells with barcodes that showed high DHFR expression beforetreatment that became resistant and survived MTX pressure. Thisindicates that the population of resistant cells expressing high DHFR(though extra copies on ecDNA) were pre-existing. Alternatively, theidentification of cells that did not have high DHFR expression beforetreatment, but show high expression of DHFR following treatmentindicates a de-novo generation of ecDNA (FIG. 10 ).

Barcoding with Parallel Resistance Replicates.

The initial naïve population of HeLa cells were barcoded by stablelentiviral-mediated integration of a unique barcode sequence into thegenome of each cell thus generating around 200,000 uniquely barcodedHela cells. This barcoded population of cells were expanded and dividedinto 8 separate resistance experiments in parallel. The cells in theseparallel replicates were treated with 100 nM MTX for several weeks togenerate resistant populations of cells. Then, each replicate ofresistant population of cells was sequenced to determine which barcodesbecame resistant. Common barcodes were identified in resistant cellsacross replicates, thereby indicating that these cells harboredresistance before treatment due to the presence of pre-existing ecDNA.In addition, a portion of barcodes were unique to individual replicates,indicating that resistance was formed de novo. (FIG. 11 ).

Example 3: Treatment of KRAS Mutant Tumors in Mice

Mice were implanted with CT26WT E3 G12C KRAS mutant tumor cells. Oncetumors reached an average volume of 350 mm³, mice were started on one ofthe following therapeutic regimens using a KRAS inhibitor (adagrasib)and/or an RNR inhibitor (gemcitabine): (1) vehicle only; (2) KRASi(adagrasib) 50 mg/kg orally once per day; (3) RNR (gemcitabine) 10 mg/kgintraperitoneal every other day; (4) RNRi (gemcitabine) 120 mg/kgintraperitoneal once per week; (5) RNRi (gemcitabine) 10 mg/kgintraperitoneal every other day+KRASi (adagrasib) 50 mg/kg orally onceper day; or (6) RNRi (gemcitabine) 120 mg/kg intraperitoneal once perweek+KRASi (adagrasib) 50 mg/kg orally once per day.

As a single agent, only KRASi (adagrasib) resulted in a significantdelay in tumor growth. However, by day 14, the tumors began to exhibitresistance to the KRASi (adagrasib) and tumor growth resumed. When theKRASi (adagrasib) was combined with the RNR inhibitor (gemcitabine),tumor growth was inhibited and continued through study day 30. Tofurther assess the effect of combination, four mice that developedresistance on the KRASi (adagrasib) treatment were switched to treatment5. Tumor growth in these mice was inhibited as compared to the mice thatremained on the single agent treatment. Data illustrating the results ofthese experiments is provided in FIG. 15 .

ecDNA were measured in metaphase spreads prepared from ex vivo culturesestablished from tumors taken from the mice on day of sacrifice. ecDNAcounts were determined using FISH for murine KRAS. As shown in FIG. 16 ,no KRAS amplified ecDNA was seen in treatments 1 or 3. In comparison,treatments that resulted in KRASi resistance accumulated high levels ofKRAS ecDNA, either at treatment group termination (D21) or on D16. KRASecDNA levels were significantly lower for mice treated with thecombination of the KRAS inhibitor and RNR inhibitor from tumors isolatedon D16.

Example 4: Treatment of KRAS Mutant Tumors in Mice

Mice were implanted with CT26WT E3 G12C KRAS mutant tumor cells. Oncetumors reached an average volume of 350 mm³, mice were started on one ofthe following therapeutic regimens using a KRAS inhibitor (adagrasib)and/or an RNR inhibitor (gemcitabine): (1) vehicle only; (2) KRASi(adagrasib) 50 mg/kg orally once per day; (3) RNRi (gemcitabine) 20mg/kg intraperitoneal every other day; (4) RNRi (gemcitabine) 10 mg/kgintraperitoneal every other day+KRASi (adagrasib) 50 mg/kg orally onceper day; or RNRi (gemcitabine) 20 mg/kg intraperitoneal every otherday+KRASi (adagrasib) 50 mg/kg orally once per day.

As shown in FIG. 17 , substantial tumor growth was seen in treatments1-3, with single agent KRASi (adagrasib) providing delay in tumor growthfor about 2 weeks before the tumors developed resistance and tumorgrowth accelerated. The combination of KRASi and RNRi significantlyinhibited tumor growth, reducing the tumor volume to near zero. At thelower dose of RNRi (treatment 4), 7 of 8 mice had a complete responseand 7 of 7 mice had a complete response at the higher dose of RNRi(treatment 5). Survival plots for the treatments in this study are shownin FIG. 18 .

Metaphase spreads were prepared from metaphase arrested and fixed exvivo cultures established from tumors taken from mice in treatmentgroups and ecDNA was visualized by FISH for murine KRAS. KRAS amplifiedecDNA was quantified by manual counts and/or by validated computeralgorithm ecSEG (software package developed based on the methods ofRajkumar et al., Semantic Segmentation of Metaphase Images ContainingExtrachromosomal DNA, iScience, Volume 21, 22 Nov. 2019, p 428-435).

As shown in FIG. 19 , tumors from vehicle treated and RNRi treated mice(treatment groups 1 and 3), little to know ecDNA was observed (92/100vehicle; 78/95 RNRi). Where ecDNA were present, the copy number was low,1-3 per metaphase spread. In contrast, KRASi treated mice (treatmentgroup 2), samples taken from the rapidly growing tumors showed a highprevalence of ecDNA (117/161 metaphase spreads having ecDNA or ecDNA andchromosomal amplification of KRAS), with a significantly higher ecDNAcount (an average of 16 KRAS ecDNA in ecDNA containing metaphasespreads). ecDNA was assessed in several mice from treatment groups 4 and5, which were removed from treatment after day 40. As expected, very lowecDNA was present in these tumors and where ecDNA was present, the copynumber was 1-7 ecDNA per metaphase spread. This data reinforces themechanism of action of RNRi as an anti-ecDNA therapeutic.

Example: 5: Ex Vivo Propagation of Resistant Tumor Cells Maintains KRASecDNA Providing Resistance In Vitro

Cells from a CT26WT E3 G12C KRAS mutant tumor that became resistant toKRASi were propagated in vitro in the presence of 1 μM KRASi(adagrasib). The cells continued to grow in the presence of the drug,confirming drug resistance. The parental CT26 WT E3 line remainedsensitive to the drug in culture. ecDNA were observed by FISH imagingand measured by manual counts and ecSEG. As shown in FIG. 20 , the drugresistant cells had high-levels of ecDNA, whereas the parental line didnot exhibit measurable ecDNA.

Example 6: KRASi and RNRi Inhibition of Tumor Growth In Vivo

KRASi resistant tumor cells cultured ex vivo from Example 5 wereimplanted into NOD-SCID mice. All mice in groups B and C were treatedwith KRASi (adagrasib) starting from the day of implantation. Whentumors reached an average of 200 mm³, the mice in group C were put ontothe combination treatment. Treatment groups are as follows: (A) vehicleonly; (B) KRASi (adagrasib) 50 mg/kg orally once per day; or (C) RNRi(gemcitabine) 20 mg/kg intraperitoneal every other day+KRASi (adagrasib)50 mg/kg orally once per day. As shown in FIG. 21 , the combination ofKRASi and RNRi inhibited tumor growth in the animals.

The implantation was repeated with similar conditions to test theefficacy of treatment with RNRi alone and in combination with KRASi.Mice were implanted with KRAS-resistant tumor cells as above. When thetumors reached 290 mm³, mice were treated the following day as follows:(A) KRASi (adagrasib) 50 mg/kg orally once per day; (B) RNRi(gemcitabine) 10 mg/kg intraperitoneal every other day; or (C) RNRi(gemcitabine) 10 mg/kg intraperitoneal every other day+KRASi (adagrasib)50 mg/kg orally once per day. Both RNRi alone and in combination withKRASi inhibited tumor growth in mice, as shown in FIG. 22 .

The experiment was repeated with similar conditions to test the efficacyof treatment with CHK1i in combination with KRASi. Mice were implantedwith the KRAS-resistant tumor cells as above. When the tumors reachedabout 180 mm³, mice were treated as follows: (A) KRASi (adagrasib) 50mg/kg orally once per day; (B) CHK1i (prexasertib) 20 mg/kg subcutaneoustwice a day for three days of every 7 days; or (C) RNRi (gemcitabine) 10mg/kg intraperitoneal every other day+KRASi (adagrasib) 50 mg/kg orallyonce per day. Both CHK1i and RNRi in combination with KRASi inhibitedtumor growth in the mice (see FIG. 23 ).

The experiment was also repeated with similar conditions to test theefficacy of treatment with WEE1i in combination with KRASi. Mice wereimplanted with the KRAS-resistant tumor cells as above. When the tumorsreached about 180 mm³ mice were treated as follows: (A) KRASi(adagrasib) 50 mg/kg orally once per day, (B) WEE1i (adavosertib) 60mg/kg orally once per day, (C) WEE1i (adavosertib) 60 mg/kg orally onceper day+KRASi (adagrasib) 50 mg/kg orally once per day, or (D) WEE1i(PD0166285) 0.3 mg/kg intraperitoneally once per day. Both of the WEE1iin combination with the KRASi inhibited tumor growth in the mice, aswell as the WEE1i, adavosertib, alone, though the most inhibition wasobserved with adagrasib and adavosertib (see FIG. 45 ).

Example 7: Treatment of SNU16 Cells In Vitro

SNU16 cells (human stomach undifferentiated adenocarcinoma cell line(ATCC)) in metaphase were assayed for the presence of MYC and FGFR2 byFISH. ecDNA was also quantified. As shown in FIG. 24 , high levels ofecDNA containing MYC and FGFR2 are present in these cells, with a subsetof the ecDNA containing both MYC and FGFR2.

The SNU16 cells were treated with 1 μM of an FGFR inhibitor(infigratinib) for 9 weeks. DNA was collected at days 3, 14, 28, 42, 56,and 63 days and qPCR was performed to assess copy number for MYC. FGFR2,and EGFR at each time point. Cells after 8 weeks of infigratinibtreatment and untreated SNU16 control cells were also assayed by FISHfor EGFR. As shown in FIG. 24 , as cells developed resistance to theFGFR inhibitor (and thus continued to survive in infigratinib), the copynumber of EGFR increased, whereas copy number for MYC and FGFR2, whichwere already amplified in the starting SNU16 cells, remained relativelyconstant. As shown by FISH in FIG. 24 , EGFR amplification localized toecDNA in these treated cells.

To assess ecDNA copy number dynamics throughout the development ofinfigratinib resistance, DNA was extracted using the QIAamp DNA Mini Kit(Qiagen) and the DNA was amplified via quantitative PCR (qPCR) usingTaqman copy number assays (ThermoFisher). The EGFR Taqman assay ID wasHs00997424_cn; GFGR2 assay ID was Hs05182482_cn; and the MYC assay IDwas Hs03660964_cn. The cycle threshold values were normalized to theinternal RNase P Taqman assay, and gene copy number was calculated usingthe ΔΔCt method and the DNA from a diploid control cell line, DLD1.

To collect cells in metaphase for fluorescent in situ hybridization(FISH), the basic protocol as described in Turner et al, 2019, wasfollowed. Briefly, cells were incubated for at least three hours withcolcemid, followed by treatment with a potassium chloride hypotonicsolution, and fixation using Camoy's solution (3:1 methanol:glacialacetic acid v/v). Fixed cells in metaphase were dropped onto humidifiedslides, followed by dehydration in ascending ethanol series. FISH probeshybridizing to EGFR, FGFR2, and MYC were purchased from Empire Genomics.Following probe hybridization, slides were washed with a solution of0.4×SSC/0.3% IGEPAL buffer, followed by a final wash in 2×SSC/0.1%IGEPAL. Mounting media containing DAPI was applied to the slide, acoverslip was added, and cells in metaphase were imaged using a KeyenceBZ-X800 microscope at 630× total magnification.

Images from the FISH assays were used to quantify the numbers of ecDNAcontaining EGFR, FGFR2, or MYC. Images were uploaded into the ecSEGsoftware developed by Boundless Bio, Inc.

Example 8: RNRi Prevents Development of Primary and Secondary Resistancein SNU16 Cells

FIG. 25 shows that simultaneous inhibition of FGFR2 with infigratiniband ecDNA with RNR inhibitor gemcitabine completely inhibits developmentof primary resistance to infigratinib, 2×10⁶ SNU16 cells were dividedinto 6 groups and treated with 1 uM infigratinib, 1 uM erlotinib, 10 nMgemcitabine, 1 uM infigratinib+1 uM erlotinib, and 1 uM infigratinib+10nM gemcitabine and the cell growth was recorded over 12 weeks. Eachtreatment arm was terminated once the cell number reached 5×10⁶.Erlotinib treatment had no effect on cell growth on its own. Gemcitabinedelayed cell growth for the first two weeks, but the cells continued togrow quickly after that. Infigratinib strongly suppressed cell growthfor 5 weeks, after which cells developed resistance and grew rapidly.Combination treatment of erlotinib and infigratinib had a strong growthinhibitory affect for the first 5 weeks, after which cells started todevelop resistance and increased their growth rate for the remainder ofthe study. However, combination treatment with infigratinib andgemcitabine completely inhibited cell growth for the entire duration ofthe study and no cells were able to develop resistance.

FIG. 26 shows that RNR inhibitor prevent development of secondaryresistance to erlotinib in SNU16 cells. The infigratinib-resistant SNU16cells (generated as shown in FIG. 25 ) were subdivided into six groupsand each group of 1×10⁶ cells was subjected to treatment with 1 uMinfigratinib, 1 uM erlotinib, 10 nM gemcitabine, or 1 uM erlotinib+10 nMgemcitabine and the cell growth was recorded over 5 weeks. Eachtreatment arm was terminated once the cell number reached 5×10⁶.Infigratinib treatment had no effect on cell growth on its own.Gemcitabine, as well as erlotinib, delayed cell growth for the first twoweeks, but the cells continued to grow quickly after that. Combinationtreatment of erlotinib and infigratinib had a moderate growth inhibitoryaffect for the first 3 weeks, after which cells started to developresistance and increased their growth rate for the remainder of thestudy. However, combination treatment with erlotinib and gemcitabinecompletely inhibited cell growth for the entire duration of the studyand no cells were able to develop resistance.

FIG. 30 shows that WEE1 inhibitor, adavosertib, prevented resistanceformation upon infigratinib treatment in SNU16 cells. SNU16 cells weresubdivided into five groups and each group of 1×10⁶ cells was subjectedto treatment with 1 μM infigratinib, 1 μM erlotinib, 1 μM infigratiniband 1 μM erlotinib, 0.1 μM adavosertib and cell growth was recorded over9 weeks. Infigratinib resulted in transient growth suppression anderlotinib treatment had no effect on cell growth on its own. Combinationtreatment of infigratinib and erlotinib delayed cell growth for aboutsix weeks but the cells continued to grow quickly thereafter. However,combination treatment with infigratinib and adavosertib completelyinhibited cell growth for the entire duration of the study and no cellswere able to develop resistance.

FIG. 34 shows that PARG inhibitor PD00017273 significantly delaysresistance formation upon infigratinib treatment in SNU16 cells. SNU16cells were subdivided into five groups and each group of 1×10⁶ cells wassubjected to treatment with infigratinib, erlotinib, PD00017273,infigratinib and erlotinib, or infigratinib and PD00017273 and cellgrowth was recorded over 9 weeks. PD00017273 and erlotinib alone hadlittle to no effect on cell growth, whereas infigratinib demonstratedtransient growth suppression for approximately 3 weeks. Combinationtreatment of infigratinib and erlotinib delayed cell growth for aboutsix weeks but the cells continued to grow quickly thereafter. However,combination treatment with infigratinib and PD00017273 reduced cellgrowth and kept it at low levels for the duration of the study.

Methods: SNU16 control cells were cultured in RPMI-1640 media with 10%FBS under standard tissue culture conditions at 37° C. and with 5% CO₂.Low passage control cells were treated with 1 μM infigratinib over thecourse of 9 weeks. Cells were passaged as needed and media andinfigratinib were replaced at least once per week. DNA was collected atday 3 and weeks 2, 4, 6, 8 and 9. Cells in metaphase were collected at 8weeks from both control and infigratinib-resistant cells.

SNU16 (CRL-5974) cell line was purchased from ATCC. SNU16 cells weregrown in RPMI 1640 medium (Fisher Scientific) with 10% FBS and 100 U/mlpenicillin/streptomycin (Fisher Scientific). For the resistanceexperiments, SNU16 cells were plated at 2 million per T75 flask and weretreated with either 1 μM infigratinib (Selleck Chemicals), 1 μMerlotinib (Selleck Chemicals), 10 nM gemcitabine (Sigma Aldrich), or acombination. The media was changed and fresh drug was added at leastonce per week. The cells were counted over the span of several weeks tomeasure cell growth.

Example 9: WEE1 Guide Dropout in HeLa ecDNA+ vs. HeLa ecDNA− KinomeCRISPR Screen

FIG. 33 shows increased sensitivity of HeLa ecDNA+ over HeLa ecDNA−cells to WEE1 knockout in a pooled CRISPR screen. Levels of normalizedsgRNA guide drop score for 10 different guides targeting WEE1 are shownfor HeLa ecDNA− cells (WT) as well as HeLa ecDNA+ cells that harbor DHFRamplified on ecDNA. HeLa ecDNA+ were grown either in the absence (−MTX)or presence (+MTX) of 1 uM methotrexate for the duration of the CRISPRscreen. Guide drop-out score relative to original levels within thepooled guide library were determined for all three arms by NGSsequencing at 7 days, 14 days and 21 days. Dotted horizontal linesdenote differential drop score for the most effective WEE1 guides inecDNA+ lines vs. ecDNA− line indicating higher sensitivity of ecDNA+lines to WEE1 knockout further supporting increased sensitivity of thesecells to inducers of replication stress.

Example 10: Basal Replication Stress Markers are Elevated andSensitivity to RS Inducers is Enhanced in ecDNA+ Cells

FIG. 38 shows ecDNA+ cells have heightened levels of basal replicationstress. To determine intrinsic single-strand DNA (ssDNA)-damage inducedreplication stress, hyperphosphorylated form of Replication Protein A(RPA) 32 Ser4/Ser8 was detected by immunofluorescence in untreated andfixed ecDNA+ (Colo320 DM) and an ecDNA− (Colo320 HSR) cell line models.Representative box and whisker plot indicating % cell quantification ofthe total p-RPA32 S4/S8 foci (minimum threshold of >3 puncta/cell)detected by immunofluorescence in ecDNA+ and ecDNA− cells. Total numberof 5000 ecDNA− and 6000 ecDNA+ cells with >3 puncta/cell were processedand analyzed. Statistical significance was calculated usingnonparametric t-test. ecDNA+ cells are found to have heightened basalreplication stress.

FIGS. 39A-39B shows that ecDNA+ cells have intrinsically reduced DNAreplication fork speeds versus ecDNA− cells with comparable MYC geneamplification on chromosomes. In FIG. 39A, to allow analysis ofreplication tracts including measurements of fork speed, DNA fiberanalysis was conducted in untreated ecDNA+ (Colo320 DM) andecDNA−(Colo320 HSR) cell line models. The two cell models werepulse-labeled with the thymidine analogues chlorodeoxyuridine (CldU) andiododeoxyuridine (IdU) for 30 min each. Cells were lysed, DNA fibersspread out and immunostained using specific antibodies against CldU andIdU. As shown in FIG. 39B, ecDNA bearing Colo320 DM illustrated averagereduced fork speed when compared with Colo320 HSR ecDNA− cells. Adifference of −0.18 kb/min in replication fork speed between ecDNA+ andecDNA− cells was observed. Statistical significance was calculated usingnonparametric Kolmogorov-Smirnov test.

FIGS. 40A-40C show inhibition of RNR blocks nucleotide synthesis causingenhanced replication stress and reduced cellular transformation inecDNA+ compared to ecDNA-tumor cells. As demonstrated, the exacerbatedreplication stress observed in ecDNA+ COLO320-DM cells upon inhibitionof the RNR results in alteration of cell-proliferation/transformation inFIG. 40A. Data shown in FIG. 40B shows a representative FISH images andquantification of changes in ecDNA carrying amplified oncogene counts.Data shown in FIG. 40C demonstrates that the effects on replicationstress by the RNRi are a result of nucleotide depletion. In FIG. 40A, toevaluate cellular transformation, an in vitro soft agar colony formationassay was conducted wherein ecDNA bearing COLO320-DM and SNU16 cellmodels were grown alongside ecDNA− cell models, COLO320-HSR and DLD1 inthis 3D-format assay for 21 days. Cells were treated individually onlyonce on the day of the seeding of cells at increasing doses of RNRi,(5-chloro-2-[[rac-(1S,2R)-2-(6-fluoro-2,3-dimethyl-phenyl)-1-(2-oxo-3H-1,3,4-oxadiazol-5-yl)propyl]sulfamoyl]benzamide).In FIG. 40B, to determine change in ecDNA carrying MYC amplifiedoncogene counts, ecDNA+ (COLO320-DM) cells were treated with an IC-90dose (10 uM) of RNRi for 21 days. Cells in log-growth phase werearrested in metaphase. FISH (Florescent in-situ hybridization) for MYConcogene was performed on the fixed metaphase spreads and the nucleiwere counterstained with DAPI. In FIG. 40C, to determine if theexacerbated replication stress observed in the ecDNA+ COLO320-DM cellsupon inhibition of the RNR is due to depleted cellular nucleotide pools,ecDNA+ COLO320-DM cells were simultaneously treated with a single IC-70dose (7 uM) of RNRi, and increasing doses of exogenous nucleosides(ATGUC) for 16 hr. Cells were lysed and immunoblotted with pRPA32-S33antibody as a marker of ssDNA-damage induced replication stress.Inhibition of RNR was found to block nucleotide synthesis causingenhanced replication stress and reduced cellular transformation inecDNA+ compared to ecDNA(−) tumor cells.

FIGS. 41A-41B show ecDNA+ cells demonstrate enhanced replication stressin response to CHK1i across multiple models. As shown in FIG. 41A, HeLaecDNA+ cells show increased sensitivity over HeLa ecDNA− cells to Chk1inhibition by GDC0575. DHFR protein levels were markedly upregulated inHeLa ecDNA+ cells which harbor DHFR ecDNA and were grown in the presenceof 1 uM methotrexate (MTX) to maintain resistance (MTX-R). Upontreatment with 200 nM GDC0575 for 24 h, HeLa ecDNA+ cells exhibited amuch stronger induction of markers of RS and DNA damage than ecDNA−cells. Increased Chk1 phosphorylation at S345 indicates higheractivation of RS-activated ATR kinase, while increased gH2AX indicatesincreased damage resulting from DNA double stranded breaks triggered byreplication stress. FIG. 41B shows an experiment to determinesingle-strand DNA (ssDNA)-damage induced replication stress uponinhibition of CHK1 pathway. Hyperphosphorylated form of ReplicationProtein A (RPA) 32 Ser4/Ser8 was detected by immunofluorescence in fixedecDNA+ (Colo320 DM) and ecDNA− (Colo320 HSR) cell line models that werepreviously treated with CHK1 i GDC-0575 for 16 hr. Representative bargraph represents % total nuclei positive for P-RPA32 S4/S8 foci (minimumthreshold of >3 puncta/nuclei) detected by immunofluorescence in ecDNA+and ecDNA− cells. CHK1 inhibition was found to increases pRPA levels inecDNA+ (DM) cells vs. matched ecDNA− (HSR) cells.

FIGS. 42A-42B show intrinsic replication fork dysfunction in ecDNA(+)cancer cells is further compromised by RNR inhibition relative toecDNA(−) cells. To allow analysis of replication tracts upon inhibitionof RNR pathway, including measurements of fork speed, DNA fiber analysiswas conducted in ecDNA+ (Colo320 DM) and ecDNA−(Colo320 HSR) cell linemodels treated with RNRi gemcitabine for 16 hr. The two cell models werethen pulse-labeled with the thymidine analogues chlorodeoxyuridine(CldU) and iododeoxyuridine (IdU) for 30 min each (FIG. 42A). Cells werelysed, DNA fibers spread out and immunostained using specific antibodiesagainst CldU and IdU. As shown in FIG. 42B, the intrinsic replicationfork dysfunction in ecDNA+ cancer cells is further compromised upon RNRinhibition relative to ecDNA− cells, although a decrease in replicationfork speed was also observed in Colo320 HSR ecDNA− cells upon treatmentwith RNRi. A difference of −0.13 kb/min in replication fork speedbetween ecDNA+ and ecDNA− cells treated with RNRi was observed.Statistical significance was calculated using nonparametricKolmogorov-Smirnov test.

FIGS. 43A-43B show intrinsic replication fork dysfunction in ecDNA+cancer cells is further compromised by CHK1 inhibition relative toecDNA− cells. To allow analysis of replication tracts upon inhibition ofCHK1 pathway including measurements of fork speed, DNA fiber analysiswas conducted in ecDNA+ (Colo320 DM) and ecDNA− (Colo320 HSR) cell linemodels treated with CHK1i Prexasertib for 16 hr. The two cell modelswere then pulse-labeled with the thymidine analogues chlorodeoxyuridine(CldU) and iododeoxyuridine (IdU) for 30 min each (FIG. 43A). Cells werelysed, DNA fibers spread out and immunostained using specific antibodiesagainst CldU and IdU. As shown in FIG. 43B, the intrinsic replicationfork dysfunction in ecDNA(+) cancer cells is further compromised uponCHK1 inhibition relative to ecDNA(−) cells, although a decrease inreplication fork speed was also observed in Colo320 HSR ecDNA− cellsupon treatment with CHK1i. A difference of −0.1 kb/min in replicationfork speed between ecDNA+ and ecDNA− cells treated with CHK1i wasobserved Statistical significance was calculated using nonparametricKolmogorov-Smirnov test.

FIG. 44 shows that targeted therapy using pharmacological inhibition ofthe primary oncogenic driver in cancer cells results in reduced proteinlevels of RRM2 subunit of RNR. Four cell lines shown that havepreviously been determined to harbor oncogenes amplified either on ecDNAor HSR, or to lack amplifications (CT26). Inhibition of the oncoproteinresulted in a rapid reduction of RRM2 protein levels within 24-48 hsuggesting that these inhibitors may indirectly lead to a reduction ofdNTPs levels and an induction of replication stress. Consistent withthis hypothesis, inhibition of ecDNA-amplified HER2 and FGFR2 in H2170and SNU16 cells, respectively, showed a concomitant increase in gH2AX, aknown marker of collapsed replication forks and DNA damage.Interestingly, KATOIII cells that harbor FGFR2 amplification as HSR, andCT26 cells that do not harbor any oncogene amplification, did not showan increase in gH2AX despite reduced RRM2 levels, further supportingthat ecDNA-bearing cells are particularly sensitive to a reduction ofRRM2 levels and replication stress. In addition, this reduction of RRM2levels in SNU16 cells was shown to be maintained for a prolonged periodof 5-6 weeks suggesting a lack of compensatory mechanisms formaintaining RNR activity. These findings strongly reinforce therationale for combination of targeted therapies with RS-inducing agentsin ecDNA-bearing cancers.

Example 11: Resistance to Targeted Therapy in ecDNA+ vs. ecDNA− Cells

Table 2 (below) shows the timeline of resistance development in ecDNA+vs. ecDNA− cells. To measure growth kinetics over time, each cell linewas treated once or twice per week with targeted inhibitor against theamplified oncogene. In the case of SKBR3 and Calu-3, irbinitinib wasincreased over time, starting at 2× the relative EC50 and then increaseto 4× the relative EC50 for a total duration of 6 weeks. In the case ofH2170, irbinitinib was used at EC90 (500 nM) upfront and the cell growthwas monitored for 3 weeks, at which point the cells were growing at asimilar rate as DMSO control cells. In the case of SNU16 and KATOIII,infigratinib was added at EC90 (1 uM) and the cell growth was monitoredfor 6 and 11 weeks, respectively.

For these long-term growth curves, the EC50 of targeted therapy wasfirst determined in short term 5 day viability assays. To determine theEC50 of irbinitinib, H2170 cells were plated at 3000 cell/well in a96-well plate; SKBR3 and Calu-3 cells were plated at 3500 cells/well ina 96-well plate. All cells were dosed with irbinitinib continuously for5 days (serial dilutions ranging from 12 nM to 1 uM) along with a DMSOcontrol. EC50 curves were determined based on cell viability usingCellTiter-Glo 2.0 reagent (Promega). To determine the EC50 ofinfigratinib, SNU16 and KATOIII cells were plated at 1000 cells/well ina 96-well plate and were treated with infigratinib continuously for 5days (serial dilutions ranging from 4 nM to 1 uM) along with a DMSOcontrol. EC50 curves were determined based on cell viability usingCellTiter-Glo 2.0 reagent (Promega). Targeted therapy directed againstdriver oncogenes showed differential effects depending on the type ofoncogene amplification. Cell lines, such as H2170 and SNU16, whichharbor oncogene amplification on ecDNA exhibited a markedly betterability to gain resistance and continue to grow in the presence oftargeted therapy than cell lines, such as SKBR3, Calu-3, and KATOIII,which harbor chromosomal oncogene amplification (Table 2).

TABLE 2 Targeted Therapy Effects 3-5 day acute Tumor Amplification Drugcytotoxicity Long-term culture Line Amplification type treatment EC50(μM) growth properties H2170 ERBB2 ecDNA irbinitinib 0.110 Increasedproliferation <3 weeks SKBP3 ERBB2 Chromosomal irbinitinib 0.043 <1%viability >6 weeks Calu3 ERBB2 Chromosomal irbinitinib 0.414 Reducedproliferation (50%), >6 weeks SNU16 FGF2 ecDNA infigratinib 0 014 Nearnormal proliferation <5 weeks KATOIII FGF2 Chromosomal infigratinib0.013 <1 viability >10 weeks

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will occur to those skilled inthe art without departing from the invention. It should be understoodthat various alternatives to the embodiments described herein may beemployed. It is intended that the following claims define the scope ofembodiments and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. A method of treating an ecDNA-associated cancer in a subject comprising: administering to the subject a therapeutically effective amount of (i) a replication stress pathway agent (RSPA), wherein the RSPA is a WEE1 inhibitor, and (ii) a cancer-targeted therapeutic agent, wherein cells of the ecDNA-associated cancer have an ecDNA signature, wherein the cells of the ecDNA-associated cancer comprise an amplification of a first gene or portion thereof and the amplification is present on ecDNA, and wherein the cancer-targeted therapeutic agent is directed against a protein encoded by the first gene, thereby decreasing growth or number of the cells of the ecDNA-associated cancer in the subject.
 2. The method of claim 1, wherein the first gene comprises an oncogene, a drug-resistance gene, a therapeutic target gene, or a checkpoint inhibitor gene.
 3. The method of claim 1, wherein the subject has not been previously treated with the cancer-targeted therapeutic agent.
 4. The method of claim 1, wherein the cells of the ecDNA-associated cancer are resistant or non-responsive to a previous therapeutic agent.
 5. The method of claim 1, wherein the method prevents an increase of ecDNA in the cells of the ecDNA-associated cancer.
 6. The method of claim 1, wherein a level of oncogene amplification and/or a level of copy number variation (CNV) in circulating cells of the ecDNA-associated cancer is reduced after treatment as compared to the level of oncogene amplification and/or CNV in the circulating cells of the ecDNA-associated cancer prior to treatment.
 7. The method of claim 1, wherein the ecDNA signature is selected from the group consisting of a gene amplification; a p53 loss of function mutation; absence of microsatellite instability (MSI-H); a low level of PD-L1 expression; a low level of tumor inflammation signature (TIS); a low level of tumor mutational burden (TMB); an increased frequency of allele substitutions, insertions, or deletions (indels); and any combination thereof. 